The malaria parasite, Plasmodium falciparum, continues to devastate human health. The emergence and spread of multi-drug resistant (MDR) parasites following decades of over-use of chloroquine (CQ) is of fundamental significance to current malaria control efforts. The near-global selective sweep of the K76T- encoding mutation in the CQ resistance (CQR) gene, pfcrt, left behind an adapted genome impacting parasite growth, fitness, and a propensity for rapid MDR evolution. The long-term goal of this project is to identify the molecular components of this resistance-adapted genome. Our hypothesis is that there is an essential genetic background of CQR parasites, comprising networked gene actions that compensate for altered PfCRT function and stabilize parasite physiology and growth in the absence of drug pressure. To detect the genome-wide presence of these drug resistance adaptations, we focus on basic growth and normal cellular processes in two different parasite solutions to historical drug pressure: 1) progeny of a genetic cross that maintain a common pfcrt allele in a segregating CQR/MDR background;2) a spectrum of laboratory-selected pfcrt mutant lines in a single controlled genetic background. Our Specific Aims build upon a detailed analysis of growth and physiological traits in the absence of drug to drive a genome-wide search for the gene networks controlling key parameters of fitness. Specific Aim 1. To precisely quantify growth and physiological parameters as indicators of parasite fitness in cultured red blood cells. We will use QTL mapping of these inter- related traits to pinpoint genome regions to facilitate searches for key genes using powerful emerging comparative databases and tools. Specific Aim 2. To identify strain-specific shifts in pfcrt-mutant transcription profiles and to map expression (e)QTL controlling inheritance of expression level traits. Specific Aim 3. To identify genome structural variants associated with CQ-selection. We will determine copy number polymorphisms in genes known to be associated with drug resistance and use high resolution CGH identification of gene copy number variations, small deletions, and some SNPs throughout the genome. Formal integration of these layers of independent but complimentary whole-genome biological data highlights a novel approach to elucidate new avenues for antimalarial intervention. Project Narrative: Malaria kills more than 2 million kids in Africa and infects more than 500 million people worldwide each year. The recently completed /Plasmodium falciparum /comparative genome sequencing project at the Broad Institute provides a vast view of genetic diversity of this species;however, few methods have been developed to use this information. Our proposal uses a combination of classical genetic linkage mapping with new tools to study gene expression variation and chromosomal structural variation around the entire genome. An integration of these tools will facilitate the mapping of the molecular determinants of fitness and virulence in dangerous multi-drug resistant malaria parasites. This knowledge will lead to new avenues of attack against this disease.